This invention relates to a method and system for automatically determining the weight of starch or similar substances in various forms of simple or complex substrates.
The quantitative analysis of starch in various substrates is required for different reasons. First of all, amylaceous substances constitute one of the principal sources of energy in animal and human food; hence there is a strong interest in starch determination for nutritional studies. Secondly, those industries which use or convert starch and amylaceous products always have a need for methods to carry out quantitative analysis of these substances. Thirdly, the market value of certain products such as bran depends directly on their starch weight content, and this, too, is responsible for an interest in methods for starch determination.
Throughout the entire specification, it is to be understood that by "starch" is meant either any solid colloid insoluble in cold water having the chemical formula (C.sub.6 H.sub.10 O.sub.5).sub.n or [C.sub.6 (H.sub.2 O).sub.5 ].sub.n which is chemically identical to any polymer of glucose containing .alpha.1.fwdarw.4 and/or .alpha.1.fwdarw.6 and/or .alpha.1.fwdarw.3 linkages, whatever its origin, whether animal or vegetable, and whatever its customary name--for instance, glycogen, fecula or tapioca--or any similar product derived directly from the starch defined above--for instance, dextrins and oligoholosides. Starch, as defined above, can be a substantially pure substance (starches from wheat, corn, casara, potato, etc.) or it can be part of more complex media (for instance, grains, vegetables, oil cakes, composite feeds, etc.).
A manual method for the rapid and specific analysis of starch in different amylaceous substrates has already been proposed. This known method involves the following steps:
Step 1: If the treated sample of the substrate to be analyzed is not already in particulate form, the material must first be divided into particles, as by crushing. In general, the particles obtained should have a particle diameter of 1 mm, or less than 0.5 mm.
Step 2: A particulate sample (for instance 0.5 g), or a test sample having the particle size stipulated above--that is to say, a diameter less than 1 mm or 0.5 mm--is suspended in a suitable aqueous medium (for instance, 25 ml of water purified by ion-exchange treatment), and the starch contained in the form of grains in the particles of the sample is dispersed in the medium. These two operations are generally carried out hydrothermally, and more specifically by "starching"; that is, by bringing the mixture of aqueous medium and particulate sample to a boil for about 3 minutes and then autoclaving the latter (140.degree. C. and 2.5 kg/cm.sup.2) for about 1 hour. In this way, there is obtained a dispersion of starch in the aqueous medium.
Step 3: All or an aliquot portion of the starch dispersion is hydrolyzed enzymatically with the use of an enzymatic hydrolysis preparation comprising at least glucoamylase (coming from strains of Rhizopus delmar or Aspergillus niger) in order to convert the dispersed starch quantitatively into D-glucose, dissolved in the aqueous dispersion medium. For this purpose, the aqueous medium comprises a buffer solution (for instance, acetic acid buffer, 2-M sodium acetate) in order to bring the hydrolysis pH to between 4 and 5, and a bactericidal agent (for example, sodium ethyl mercury thiosalicylate, also known as sodium merthiolate) which is capable of inhibiting the development of any microorganism. The hydrolysis is carried out with continuous agitation at a temperature close to 50.degree.0 to 60.degree. C. for about 5 hours. The concentration by weight of the enzymatic preparation relative to the weight of the sample of substrate analyzed varies, depending on the origin and degree of purity of the hydrolysis enzyme. In general, it is between 10 and 20%, which corresponds on the average to about 150 I.U. of glucoamylase for 0.5 g of starch. A hydrolysate is thus obtained.
Step 4: From the hydrolysate, a glucose solution is prepared containing a predetermined portion, in the present case all of the glucose resulting from the hydrolysis of the starch. For this purpose, the hydrolysate is filtered with a folded paper filter and the insoluble solid residue collected on the filter is washed quantitatively, so that the filtrate obtained constitutes the desired glucose solution.
Step 5: A weight determination of the glucose contained in the glucose solution is then effected, from which there is deducted the starch content by weight of the substrate analyzed.
For this purpose, one proceeds by enzymatic and colorimetric means, first of all by oxidizing the glucose in aqueous and/or alcoholic phase so as to form gluconic acid, with liberation of hydrogen peroxide due to the catalytic action of an enzyme; namely, glucose-oxydase. Secondly, by reacting the hydrogen peroxide thus liberated with a hydrogen-donor chromogenic agent--namely, orthodianisidine--with the generation of a coloring substance due to the catalytic action of another enzyme; namely, peroxydase. Thirdly, by blocking the two reactions defined above either with hydrochloric acid, in which case one obtains a yellow color, or with sulfuric acid, in which case one obtains a pink color. The optical density of the color thus obtained, which is proportional to the amount of glucose, is then measured, from which one deduces the starch content by weight of the substrate analyzed.
The action of these enzymes is enhanced by a pH of the order of 7.2, corresponding to the introduction into the reaction medium of a buffer having a base of trihydroxymethylaminomethane, also known as TRIS buffer. The two reactions defined above require a period of time of about 45 minutes, a temperature of about 20.degree. C., and complete darkness in order to develop.
When a complete substrate contains, in addition to the starch, glucose and/or oligoholosides and/or dextrines and/or glucides capable of liberating glucose under the action of glucoamylase, the above-described analytical method does not make it possible to distinguish and determine these different substances separately. If one desires to analyze them separately, it is then necessary to have recourse to one of the following additional preliminary operations:
(a) If one wishes to determine solely the starch content, it is then necessary, prior to the dispersion (Step 1) described above in aqueous medium, to proceed with an aqueous and/or alcoholic extraction (40% alcohol) to eliminate dextrines and/or glucides and/or oligoholosides. The determination of the starch content is then carried out on the residue.
(b) If one desires to determine starch plus dextrines, it is then necessary, before the dispersion (1) described above in aqueous medium, to carry out an alcoholic extraction (80% alcohol) in order to eliminate the glucose and the other oligoholosides.
For more details concerning the method defined above and different variants thereof, reference may be had to the following publications:
(1) Article entitled "Determination of Starch in Complex Media" by P. Thivend, C. Mercier and A. Guilbot, pp. 513-526, vol. 5 (4) of the Annales de Biologic animale, Biochimie, Biophysique of 1965.
(2) Article entitled "Determination of Starch with Glucoamylase" by P. Thivend, C. Mercier and A. Guilbot, pp. 100-105, Vol. VI, of the book having the general title Methods in Carbohydrate Chemistry, edited by R. L. Whistler, Academic Press (New York and London).
(3) Article entitled "Use of Glucose Amylase for the Determination of Starch" by P. Thivend, C. Mercier and A. Guilbot, pp. 278-283, issue 9 of the 17th year of the 1965 book having the general title "Der Starke" (Wissenschaftliche Verlagagesellschaft M.B.H., Stuttgart).
(4) Publication entitled "The Enzymatic Determination of Starch in Dietary Foods" by H. Ruttloff, M. Rothe, R. Freise and R. Schierbaum, pp. 201-212, Vol. 130, issue 4 of the 1966 journal entitled Zeitschrift fur Lebensmitteln Untersuchen und Forschnung.
(5) Article by E. Y. C. Lee and W. J. Whelan, published in 1966 in the Archives of Biochemistry and Biophysics, Vol. 116, p. 162.
(6) Article by M. L. Salo and M. Salmi, entitled "Determination of starch by the amyloglucosidase method" published in 1968 in the Journal of the Scientific Agricultural Society of Finland, pp. 38-45, Vol. 40.
(7) Article by J. C. Macrae, D. C. Armstrong, entitled "Enzymatic method for determination of .alpha.-linkage glucose polymers in biological materials" published in 1968, pp. 578-581, Vol. 19, Journal of Science and Food Agriculture.
(8) Article by I. F. Ebeli, published on page 25 of Vol. 8, 1969, of Phytochemistry.
(9) Article by F. Meuser and W. Kempf, entitled "Analytical Problems and Possible Uses of Enzymatic Starch Determination," published in 1970, Vol. 22, 12 (pp. 417-423) of Die Starke.
(10) Article by R. A. Libby, entitled "Direct Starch Analysis using DMSO Solubilization and Glucoamylase," published in 1970, pp. 473-481, Vol. 47 of Cereal Chemistry.
(11) Article by R. F. H. Dekker and G. N. Richards, entitled "Determination of Starch in Plant Material," published in 1971, pp. 441-444, Vol. 22, Journal of Science and Food Agriculture.
All the articles mentioned above are incorporated by reference in the present specification.
Though the method of analysis described above has made it possible extensively to simplify the determination of starch, it nevertheless is still relatively lengthy. In practice, it is not feasible to determine more than 12 samples per day under the usual conditions which prevail in an analytical laboratory. This is due essentially to the fact that the operations of:
dispersing the starch of a particulate sample in an aqueous medium by "starching" and autoclaving, PA1 enzymatic hydrolysis, PA1 preparation of the glucose solution necessary for the titration, by filtration,
remain essentially manual operations; that is to say, each of them requires one or more personal actions to carry out.
Under these circumstances, automating the above-described prior art method of starch determination appears to be impossible. Yet there is an ever increasing need for methods which make it possible automatically to carry out a large number of serial determinations of starch content.